In this study, we functionally analyzed the gene family encoding necrosis- and ethylene-inducing-like proteins (NLPs) of the vascular wilt pathogen Verticillium dahliae. We show that the composition of the NLP gene family varies little among V. dahliae isolates. The cytotoxic activity of NLP family members of a tomato pathogenic V. dahliae strain was determined, demonstrating that only two of the seven NLPs induced plant cell death. The genes encoding these cytotoxic NLPs were found to be induced in V. dahliae upon colonization of tomato. Interestingly, targeted deletion of either of the two genes in V. dahliae significantly compromised virulence on tomato as well as on Arabidopsis plants, whereas deletion of only one of the two genes affected virulence on N. benthamiana. This could be attributed to differential induction of the two NLP genes in V. dahliae upon N. benthamiana colonization, revealing that the in planta induction of NLP genes varies between plant hosts. Intriguingly, one of the NLP genes appears to also affect vegetative growth and conidiospore production, as the corresponding deletion strain produced significantly less conidiospores and developed extensive aerial mycelium. In conclusion, we demonstrate that the expanded V. dahliae NLP family shows functional diversification, not only revealing differential cytotoxicity between family members, but also that the cytotoxic NLPs play a role in vegetative growth and asexual reproduction in addition to their contribution to virulence.